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1. Scope
1.1 This test method covers the determination of iron
bacteria by examination under the microscope. The method
provides for the identification of the following genera of
bacteria found in water and water-formed deposits: Siderocapsa, Gallionella (Dioymohelix), Sphaerotilus, Crenothrix,
Leptothrix, and Clonothrix.
1.2 This standard does not purport to address the safety
concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety and
health practices and determine the applicability of regulatory
limitations prior to use.
6. Apparatus
6.1 Centrifuge, complete with conical tubes.
6.2 Microscope that provides a magnification of 400 to
10003 and is complete with a suitable light source. A
dark-field condenser is desirable.
6.3 Pipets, Mohr-type, 10-mL, with an opening 3 to 4 mm
in diameter, for thick samples, and 1-mL Mohr-type pipets for
thin samples.
6.4 Spatula, small and narrow, for handling thick samples.
6.5 Membrane Filter, with appropriate filter-holding assembly (see 9.2).
2. Referenced Documents
2.1 ASTM Standards:
D 887 Practices for Sampling Water-Formed Deposits2
D 1129 Terminology Relating to Water3
D 1193 Specification for Reagent Water3
D 3370 Practices for Sampling Water from Closed Conduits3
3. Terminology
3.1 DefinitionsFor definitions of terms used in this test
method, refer to Terminology D 1129.
7. Reagents
7.1 Purity of ReagentsReagent grade chemicals shall be
used in all tests. Unless otherwise indicated, it is intended that
all reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society,
where such specifications are available.5 Other grades may be
used, provided it is first ascertained that the reagent is of
sufficiently high purity to permit its use without lessening the
accuracy of the determination.
7.2 Purity of Water Unless otherwise indicated, references to water shall be understood to mean reagent water
conforming to Specification D 1193, Type II.
7.3 Ammonium Oxalate-Crystal Violet SolutionPrepare
Huckers modification of the Gram stain (4) by mixing a
1
This test method is under the jurisdiction of ASTM Committee D-19 on Water
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved Aug. 30, 1985. Published October 1985. Originally
published as D 932 47 T. Last previous edition D 932 72 (1984)e 1.
2
Annual Book of ASTM Standards, Vol 11.02.
3
Annual Book of ASTM Standards, Vol 11.01.
4
The boldface numbers in parentheses refer to the list of references at the end of
this test method.
5
Reagent Chemicals, American Chemical Society Specifications, Am. Chemical Soc., Washington, DC. For suggestions on the testing of reagents not listed by
the American Chemical Society, see Reagent Chemicals and Standards, by Joseph
Rosin, D. Van Nostrand Co., Inc., New York, NY, and the United States
Pharmacopeia.
Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D 932 85 (2002)
TABLE 1 Key for Identification of Bacteria
D 932 85 (2002)
D 932 85 (2002)
9.4 Place the HCl (1 + 4) at one side of the cover glass and
draw it underneath by absorbing the liquid at the opposite side
by means of a filter paper or blotter. Continue this procedure
until no more yellow ferric chloride is evident in the solution.
Take care that the flow of the liquid is not fast, or the sample
may be drawn to the absorbent material. This treatment
removes the iron deposited in the sheaths of the bacteria and
allows the cells to be seen.
9.5 In a similar manner, rinse the iodine solution under the
cover glass until the color of the liquid becomes yellow or the
filter paper becomes colored. The iodine stains the bacterial
cells brown and makes them more easily visible.
9.6 Examine the slide under a microscope, using a highpower, dry objective, for the presence of Sphaerotilus, Crenothrix, Leptothrix, and Clonothrix. If used carefully, an oilimmersion lens may be helpful.
9.7 Prepare a new slide by placing a drop of the sample on
a clean slide and allowing it to air-dry. Then stain it for 1 min
with ammonium oxalate-crystal violet solution, wash it with
water, and allow it to dry. Examine the slide under anoilimmersion lens for the presence of Siderocapsa, which will
appear violet colored.
10. Report
10.1 The report shall state Present or Not found, probably absent. Make a statement as to the relative abundance of
the organisms present. Make a negative report only after
examination of several slides.
D 932 85 (2002)
FIG. 6 Crenothrix polyspora. Cells enclosed within a sheath of ferric hydrate and showing false branching. Magnification about 390 3 .
Plate 3, Fig. B of Ref (8)
D 932 85 (2002)
D 932 85 (2002)
FIG. 9 Clonothrix ferruginea. Sketch showing cells enclosed within sheath and
false branching. Magnification about 430 3 . Fig. 4 of Ref (7)
D 932 85 (2002)
FIG. 10 Crenothrix polyspora. Conidia can be seen inside and coming out at ends of filaments. Magnification about 345 3 . Fig. 5 of Ref
(9)
REFERENCES
(1) Bergey, D. H., Manual of Determinative Bacteriology, 8th Edition,
Williams & Wilkins Co., Baltimore, MD, 1974.
(2) Salle, A. J., Fundamental Principles of Bacteriology, McGraw-Hill
Book Co., Inc., New York, NY, 1943, pp. 516519.
(3) Starkey, R. L., Transformation of Iron by Bacteria in Water, Journal
of the American Water Works Association, Vol. 37, 1945, pp. 963984.
(4) Manual of Methods for Pure Culture Study of Bacteria, Biotech
Publications, Geneva, NY, 1946, Chapter IV, pp. 4648.
(5) Hardman, Yvette, and Henrici, A. T., Studies of Fresh Water Bacteria
V. Distribution of Siderocapsa treubii in Some Lakes and Streams,
Journal of Bacteriology, Vol 37, 1939, p. 97.
(6) Mitchell, R., Water Pollution Microbiology Vol. 1, Wiley-Interscience,
New York, NY, 1972.
(7) Standard Methods for the Examination of Water and Waste Water.
American Public Health Assoc., 19th Edition, 1976, p. 1000.
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